Abstract
Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology. Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.
Highlights
Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology
Distinct cell populations are labeled with unique signatures of amine-reactive fluorescent dyes of different emission wavelengths and intensities before pooling for exposure to potentially reactive antibody followed by analysis by flow cytometry
A basal cell line was stably barcoded with unique combinations of four endogenous fluorescent proteins (FPs) to generate a stable and adaptable panel of 16 distinct reporter cell lines
Summary
Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology. We describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. In this proofof-concept study, we established a 16-plex basic panel and tested the feasibility of expanding to a 256-plex panel of reporter cell lines We successfully applied this platform to multiplex detection of antibody binding to cell panels expressing various influenza A hemagglutinin (HA) trimers or human CCR2b and CCR5, and several domain-swap or point mutants that define specific protein domains/epitopes
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